1) Field of the Invention
This invention relates to methods for the reduction of cross-talk in multiplex capillary electrophoresis.
2) Description of Related Art
Capillary electrophoresis (CE) instruments use electric fields to separate molecules within narrow-bore capillaries (typically 20-100 μm internal diameter). By applying electrophoresis in a small diameter fused silica capillary column carrying a buffer solution, the sample size requirement is smaller and the speed of separation and resolution is significantly higher relative to slab gel-electrophoresis. UV absorption and laser induced fluorescence are routinely used as the detection system for CE separation.
CE techniques are employed in numerous applications, including DNA sequencing, nucleotide quantification, mutation/polymorphism analysis, SDS-protein separation, and carbohydrate analysis. In order to improve sample throughput, multiple capillaries or channels are used to perform separations in parallel. For example, in one system a beam expander and a cylindrical lens are used to distribute laser light into a thin line that intersects the axes of the capillaries to provide laser induced fluorescent detection for a multiplexed CE system (K. Ueno et al., Anal. Chem., 66, 1424 (1994)). U.S. Pat. No. 5,582,705 used a laser as the excitation light source for fluorescence detection for a multiplexed CE system, while U.S. Pat. No. 6,788,414 revealed a method to perform UV absorption detection in a multiplexed CE system.
With all of the capillaries or channels illuminated at the same time, scattering, refraction, or reflection of light from neighboring channels will affect the detected channel. That is, detection in one capillary can be influenced by light absorption or fluorescence in the adjacent capillaries, thus affecting trace analysis. This phenomenon is referred to as cross-talk between adjacent capillaries. Cross-talk in the range of 1% to 10% and even higher can be observed in the previously mentioned inventions. For accurate analysis, cross-talk needs to be eliminated if possible.
There is therefore a need to reduce or eliminate the potentially negative cross-talk effects for trace analyte detection using CE.
There are several prior art patented techniques to overcome the cross-talk in multiplex capillary systems. For example, U.S. Pat. No. 5,274,240 used a mechanical stage to translate the capillary bundle to observe one capillary at a time. U.S. Pat. No. 5,324,401 used individual optical fibers to collect emission light from each capillary to eliminate cross-talk. U.S. Pat. No. 5,790,727 used a waveguide to collect the fluorescent signal to reduce cross-talk. Although these various implementations of different optical designs in the hardware to reduce the cross-talk are effective, the cost and the complication of these hardware designs are high. There is thus a need to develop simpler less expensive alternate methods of eliminating cross-talk without increasing instrument complexity or cost. This invention has its primary objective fulfilling this need.